DESCRIPTION: (applicant's abstract) Vitamin B12 is an essential human nutrient required as a coenzyme for adenosylcobalamin dependent methylmalonyl-CoA mutase and methylcobalamin dependent methionine synthase. In addition to the B12-dependent enzymes there are a number of B12 transport proteins involved in the regulation and delivery of cobalamins. The key catalytic element in B12 is a unique carbon-cobalt bond. The aim of the present research program is to use time-resolved spectroscopy to elucidate the role of the protein in controlling and directing the reactivity of the carbon-cobalt bond. The B12 enzymes comprise a class of protein systems which lend themselves beautifully to the application of time-resolved spectroscopic techniques. Excitation with visible light results in cleavage of the C-Co within a few picoseconds. The availability of crystal structures for the B12 binding domain of two enzymes and the ability to overexpress and produce significant amounts of various wild-type and mutant enzymes makes the application of physical methods to the study of mechanism in B12 particularly promising. The work proposed will investigate the photochemical and photophysical properties of the carbon-cobalt bond in alkylcobalamins and alkylcorrinoids in solution as a function of pH and solvent, and as a function of the axial base. A thorough understanding of the free cofactors will lend insight into the ways that the labile C-Co bond is utilized and controlled in nature. This project will also involve the direct study of alkylcobalamins bound to B12 proteins, particularly methionine synthase and glutamate mutase. These studies will explore the radical caging properties of the proteins. In addition the role of the axial histidine ligand supplied by the protein will be probed by using a series of mutations to remove/replace this histidine and to alter the amino acid residues hydrogen bonded to the histidine.